KR940701454A - Determination of Polynucleotides into Selectable Cleavage Sites - Google Patents

Determination of Polynucleotides into Selectable Cleavage Sites

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Publication number
KR940701454A
KR940701454A KR1019930703922A KR930703922A KR940701454A KR 940701454 A KR940701454 A KR 940701454A KR 1019930703922 A KR1019930703922 A KR 1019930703922A KR 930703922 A KR930703922 A KR 930703922A KR 940701454 A KR940701454 A KR 940701454A
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support
polynucleotide
reagent
nucleic acid
cleavage site
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KR1019930703922A
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Korean (ko)
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에스. 어디어 마이클
혼 토마스
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로버트 피. 블랙버언
카이론 코오퍼레이션
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Publication of KR940701454A publication Critical patent/KR940701454A/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/22—Amides of acids of phosphorus
    • C07F9/24—Esteramides
    • C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/2429—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of arylalkanols
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Abstract

예정된 스트린전시에서 혼성화의 존재 또는 결여가 지지체로부터 표지의 방출을 제공하는 경우 분석물에서 관심있는 서열에 실질적으로 상동관계가 있는 올리고 뉴클레오티드 서열을 가지는 폴리뉴클레오티드를 사용하는 색산분석물을 분석하기 위한 신규한 방법이 제공된다. 특히 여러 기술이 지지체에 표지를 결합시키는데 사용되어 그 결과 지지체와 표지간의 결합의 과요드산염 절단이 표지를 방출하여 표본에서 특정 올리고 뉴클레오티드 서열의 존재를 표시하는 것으로서 검출될 수 있다.For the analysis of chromatic analytes using polynucleotides having oligonucleotide sequences that are substantially homologous to the sequence of interest in the analyte when the presence or lack of hybridization at a predetermined string provides release of the label from the support. New methods are provided. In particular, various techniques can be used to bind a label to a support such that the periodate cleavage of the bond between the support and the label can be detected by releasing the label to indicate the presence of a particular oligonucleotide sequence in the sample.

이 방법은 병의 진단, 유전인자의 기록 및 핵산혼합물의 분석에 사용한다.This method is used for diagnosis of disease, recording of genetic factors and analysis of nucleic acid mixtures.

Description

선택가능한 절단부위로의 폴리뉴클레오티드의 결정Determination of Polynucleotides into Selectable Cleavage Sites

본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음Since this is an open matter, no full text was included.

제1도는 고체지지체에 표지의 특이결합과 비특이결합사이의 차이를 설명한다.1 illustrates the difference between specific and nonspecific binding of a label to a solid support.

제2A도부터 제2D도는 선택적으로 절단가능한 부위를 분석물/프로브 복합체를 통해 지지체와 표지사이에 도입하는 본 발명의 바람직한 방법을 도식적으로 설명한다.2A to 2D diagrammatically illustrate a preferred method of the invention for introducing a selectively cleavable site through an analyte / probe complex between a support and a label.

Claims (12)

다음의 단계로 이루어진 핵산표본에 존재하는 핵산분석물에서 관심있는 올리고 뉴클레오티드 서열의 존재 검출방법: 혼성화 상태하에서 폴리뉴클레오티드 시약과 상기 핵산표본을 결합시키는 단계, 여기서 상기 표본의 하나 또는 상기 시약의 성분은 지지체에 결합되고 상기 분석물과 상기 폴리뉴클레오티드 시약의 혼성화하는 선택가능한 절단부위 -R1-O-X-O-R2-를 통해서 상기 지지체에 결합되어 있는 표지로 결과된다.A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample comprising the steps of: combining a polynucleotide reagent with the nucleic acid sample under hybridization, wherein one of the samples or a component of the reagent is The result is a label that is bound to the support and bound to the support via a selectable cleavage site -R 1 -OXOR 2 -that hybridizes the analyte with the polynucleotide reagent. 여기서 R1및 R2는 독립적으로 알킬렌, 알케닐렌, 시클로알킬렌, 시클로알케닐렌, 시클로옥시알킬렌, 아릴, 아랄킬렌 및 이것의 조합으로 이루어진 군으로부터 선택되고 X는 과요드산염-절단가능한 결합이다; 실질적으로 상기 선택가능한 절단부위를 통하는 것 외에 상기 지지체에 결합된 표지의 상기 지지체를 제거하는 단계; 과요드산염 시약으로 상기 절단부위를 절단하는 단계; 그리고 상기 지지체를 제거한 표지를 검출하는 단계.Wherein R 1 and R 2 are independently selected from the group consisting of alkylene, alkenylene, cycloalkylene, cycloalkenylene, cyclooxyalkylene, aryl, aralkylene and combinations thereof and X is a periodate-cleavable It is a bond; Removing said support of the label bound to said support in addition to substantially through said selectable cleavage site; Cutting the cleavage site with a periodate reagent; And detecting the label from which the support is removed. 제1항에 있어서, 상기 폴리뉴클레오티드 포획 프로브로 이루어지고, 상기 제1 또는 제2프로브는 상기 분석물에 존재하는 서열에 상보적인 올리고뉴클레오티드 서열을 가져 상기 혼성화 상태하에서 관심있는 서열이 있는 상기 올리고뉴클레오티드 서열의 적어도 하나와 이중체를 형성하며 상기 포획 프로브가 상기 선택가능한 절단부위를 함유하는 것을 특징으로 하는 검출방법.The oligonucleotide of claim 1, wherein said polynucleotide capture probe consists of said first or second probe having an oligonucleotide sequence complementary to a sequence present in said analyte and having said sequence of interest under said hybridization state. Forming a duplex with at least one of the sequences and wherein said capture probe contains said selectable cleavage site. 다음 단계로 이루어진 핵산 표본에 존재하는 핵산분석물에서 관심있는 올리고뉴클레오티드 서열의 존재 검출방법: 수성배지중의 혼성화 상태하에서 폴리뉴클레오티드 시약과 상기 핵산표본을 결합시키는 단계, 여기서 상기 표본의 하나 또는 상기 시약의 성분은 지지체에 결합되고 상기 분석물과 상기 폴리뉴클레오티드 시약의 혼성화는 선택가능한 절단부위 -R1-O-X-O-R2-를 통해서 상기 지지체에 결합되어 있는 표지로 결과된다. 여기서, R1및 R2는 독립적으로 알킬렌, 알케닐렌, 시클로알킬렌, 시클로알케닐렌, 시클로옥시알킬렌, 아릴, 아랄킬렌 및 이것의 조합으로 이루어진 군으로부터 선택되고 X는 과요드산염-절단가능한 결합이다; 상기 수성배지로부터 결합된 폴리뉴클레오티드 시약과 핵산분석물을 가지는 상기 지지체를 분리시키는 단계; 상기 선택가능한 절단부위를 통하는 것 외에 상기 지지체에 결합된 표지가 제거되도록 상기 수성배지로부터 다양한 혼성화 스트린전시의 배지로 상기 지지체를 세척하는 단계; 과요드산염 시약으로 상기 절단부위를 절단하는 단계; 그리고 상기 지지체를 제거한 표지를 검출하는 단계.A method for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample comprising: combining a polynucleotide reagent and the nucleic acid sample under hybridization in an aqueous medium, wherein one of the samples or the reagent The component of is bound to the support and hybridization of the analyte with the polynucleotide reagent results in a label bound to the support via a selectable cleavage site -R 1 -OXOR 2- . Wherein R 1 and R 2 are independently selected from the group consisting of alkylene, alkenylene, cycloalkylene, cycloalkenylene, cyclooxyalkylene, aryl, aralkylene and combinations thereof and X is a periodate-cut Is a possible combination; Separating the support having the bound polynucleotide reagent and nucleic acid analyte from the aqueous medium; Washing the support with a medium of various hybridizing strings from the aqueous medium to remove the label bound to the support in addition to through the selectable cleavage site; Cutting the cleavage site with a periodate reagent; And detecting the label from which the support is removed. 제3항에 있어서, 상기 폴리뉴클레오티드 시약이 지지체와 제2폴리뉴클레오티드 표지 프로브에 결합된 제1폴리뉴클레오티드 포획 프로브로 이루어지고, 상기 제1 또는 제2프로브는 상기 분석물에 존재하는 서열에 상보적인 올리고뉴클레오티드 서열을 가져 상기 혼성화 상태하에서 관심있는 서열이 있는 상기 올리고뉴클레오티드 서열의 적어도 하나와 이중체를 형성하며 상기 포획 프로브가 상기 선택가능한 절단부위를 함유하는 것을 특징으로 하는 검출방법.The method of claim 3, wherein the polynucleotide reagent consists of a first polynucleotide capture probe coupled to a support and a second polynucleotide labeled probe, wherein the first or second probe is complementary to the sequence present in the analyte. And having an oligonucleotide sequence to form a duplex with at least one of the oligonucleotide sequences having the sequence of interest under the hybridization state, wherein the capture probe contains the selectable cleavage site. 제1항에 있어서, X가The compound of claim 1 wherein X is (여기서 R은 수소 또는 알킬이다)로 이루어진 군으로부터 선택되는 것을 특징으로 하는 검출방법.Wherein R is hydrogen or alkyl. 제3항에 있어서, X가The compound of claim 3 wherein X is (여기서 R은 수소 또는 알킬이다)로 이루어진 군으로부터 선택되는 것을 특징으로 하는 검출방법.Wherein R is hydrogen or alkyl. 프로브가 지지체 한쪽 끝 기부에 결합된 폴리뉴클레오티드로 이루어지고 관심있는 상기 서열에 상보적인 서열을 가지는 반대쪽 끝이 있고, 상기 프로브가 부가적으로 선택가능 절단부위 -R1-O-X-O-R2-를 포함하는(여기서, R1및 R2는 독립적으로 알킬렌, 알케닐렌, 시클로알킬렌, 시클로알케닐렌, 시클로옥시알킬렌, 아릴, 아랄킬렌 및 이것의 조합으로 이루어진 군으로부터 선택되고 X는 과요드산염-절단가능한 결합이다) 핵산표본에 존재하는 핵산분석물에서 관심있는 올리고뉴클레오티드 서열의 존재를 검출하는데 유용한 프로브.The probe consists of a polynucleotide bound to one end of the support and has the opposite end having a sequence complementary to the sequence of interest, the probe additionally comprising a selectable cleavage site -R 1 -OXOR 2- ( Wherein R 1 and R 2 are independently selected from the group consisting of alkylene, alkenylene, cycloalkylene, cycloalkenylene, cyclooxyalkylene, aryl, aralkylene and combinations thereof and X is a periodate-cut Possible binding) A probe useful for detecting the presence of an oligonucleotide sequence of interest in a nucleic acid analyte present in a nucleic acid sample. 다음 구조를 가지는 폴리뉴클레오티드 시약.Polynucleotide reagent having the following structure. 여기서 DNA1은 DNA의 제1요소이고, DNA2는 DNA의 제2요소이고, R1및 R2는 독립적으로 알킬렌, 알케닐렌, 시클로알킬렌, 시클로알케닐렌, 시클로옥시알킬렌, 아릴, 아랄킬렌, 및 이것의 조합으로 이루어진 군으로부터 선택되고, 그리고 X는 과요드산염-절단가능한 결합이다.Wherein DNA 1 is the first element of DNA, DNA 2 is the second element of DNA, and R 1 and R 2 are independently alkylene, alkenylene, cycloalkylene, cycloalkenylene, cyclooxyalkylene, aryl, Aralkylene, and combinations thereof, and X is a periodate-cleavable bond. 제8항에 있어서, X는The compound of claim 8, wherein X is (여기서 R은 수소 또는 알킬이다)로 이루어진 군으로부터 선택되는 것을 특징으로 하는 폴리뉴클레오티드 시약.Wherein R is hydrogen or alkyl. 제8항에 있어서,The method of claim 8, 인 것을 특징으로 하는 폴리뉴클레오티드 시약.Polynucleotide reagent, characterized in that. Y1-O-R1-O-X-O-R2-O-Y2구조로써 주어진 폴리뉴클레오티드 합성에 유용한 시약. 여기서 R1및 R2는 독립적으로 알킬렌, 알케닐렌, 시클로알킬렌, 시클로알케닐렌, 시클로옥시알킬렌, 아릴, 아랄킬렌 및 이것의 조합으로 이루어진 군으로부터 선택되고, X는 과요드산염-절단가능한 결합이고, Y1은 산-민감성, 염기-안정성 보호기이고, Y2는 수소, 포스포르아미다이트, 포스포트리에스테르, 포스포디에스테르, 아인산염, H-포스포네이트 및 포스포로티오에이트로 이루어진 군으로부터 선택된다.Y 1 -OR 1 -OXOR 2 -OY useful reagents for polynucleotide synthesis given by 2 structure. Wherein R 1 and R 2 are independently selected from the group consisting of alkylene, alkenylene, cycloalkylene, cycloalkenylene, cyclooxyalkylene, aryl, aralkylene and combinations thereof, X is a periodate-cut Possible bonds, Y 1 is an acid-sensitive, base-stable protecting group, and Y 2 is hydrogen, phosphoramidite, phosphoester, phosphodiester, phosphite, H-phosphonate and phosphorothioate Selected from the group consisting of. 제11항에 있어서, 다음 구조식The structure according to claim 11, wherein (여기서 DMT는 디메톡시트리틸을 나타내고 iPr은 이소프로필을 나타낸다)을 가지는 것을 특징으로 하는 시약.Wherein DMT represents dimethoxytrityl and iPr represents isopropyl. ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.※ Note: The disclosure is based on the initial application.
KR1019930703922A 1991-06-17 1992-06-12 Determination of Polynucleotides into Selectable Cleavage Sites KR940701454A (en)

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JP2002369699A (en) 2002-12-24
WO1992022671A1 (en) 1992-12-23
CA2110591A1 (en) 1992-12-23
EP0610215A1 (en) 1994-08-17
JPH11235198A (en) 1999-08-31
IE921940A1 (en) 1992-12-30

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